Kinetic studies on the oxidation of semiquinone and hydroquinone forms of Arabidopsis cryptochrome by molecular oxygen

Cryptochromes (crys) are flavoprotein photoreceptors present throughout the biological kingdom that play important roles in plant development and entrainment of the circadian clock in several organisms. Crys non-covalently bind flavin adenine dinucleotide (FAD) which undergoes photoreduction from the oxidised state to a radical form suggested to be active in signalling in vivo. Although the photoreduction reactions have been well characterised by a number of approaches, little is known of the oxidation reactions of crys and their mechanisms. In this work, a stopped-flow kinetics approach is used to investigate the mechanism of cry oxidation in the presence and absence of an external electron donor. This in vitro study extends earlier investigations of the oxidation of Arabidopsis cryptochrome1 by molecular oxygen and demonstrates that, under some conditions, a more complex model for oxidation of the flavin than was previously proposed is required to accommodate the spectral evidence. In the absence of an electron donor, photoreduction leads predominantly to the formation of the radical FADHradical dot. Dark recovery most likely forms flavin hydroperoxide (FADHOOH) requiring superoxide. In the presence of reductant (DTT), illumination yields the fully reduced flavin species (FADH?). Reaction of this with dioxygen leads to transient radical (FADHradical dot) and simultaneous accumulation of oxidised species (FAD), possibly governed by interplay between different cryptochrome molecules or cooperativity effects within the cry homodimer

Ion mobility action spectroscopy of flavin dianions reveals deprotomer-dependent photochemistry

The intrinsic optical properties and photochemistry of flavin adenine dinucleotide (FAD) dianions are investigated using a combination of tandem ion mobility spectrometry and action spectroscopy. Two principal isomers are observed, the more stable form being deprotonated on the isoalloxazine group and a phosphate (N-3,PO4 deprotomer), and the other on the two phosphates (PO4,PO4 deprotomer). Ion mobility data and electronic action spectra suggest that photo-induced proton transfer occurs from the isoalloxazine group to a phosphate group, converting the PO4,PO4 deprotomer to the N-3,PO4 deprotomer. Comparisons of the isomer selective action spectra of FAD dianions and flavin monoanions with solution spectra and gas-phase photodissociation action spectra suggests that solvation shifts the electronic absorption of the deprotonated isoalloxazine group to higher energy. This is interpreted as evidence for significant charge transfer in the lowest optical transition of deprotonated isoalloxazine. Overall, this work demonstrates that the site of deprotonation of flavin anions strongly affects their electronic absorptions and photochemistry

Blue light induces radical formation and autophosphorylation in the light-sensitive domain of Chlamydomonas cryptochrome

Immeln D, Schlesinger R, Heberle J, Kottke T. Blue light induces radical formation and autophosphorylation in the light-sensitive domain of Chlamydomonas cryptochrome. JOURNAL OF BIOLOGICAL CHEMISTRY. 2007;282(30):21720-21728

Blue Light Induces Radical Formation and Autophosphorylation in the Light-sensitive Domain of Chlamydomonas Cryptochrome

Cryptochromes are sensory blue light receptors mediating various responses in plants and animals. Studies on the mechanism of plant cryptochromes have been focused on the flowering plant Arabidopsis. In the genome of the unicellular green alga Chlamydomonas reinhardtii, a single plant cryptochrome, Chlamydomonas photolyase homologue 1 (CPH1), has been identified. The N-terminal 500 amino acids comprise the light-sensitive domain of CPH1 linked to a C-terminal extension of similar size. We have expressed the light-sensitive domain heterologously in Escherichia coli in high yield and purity. The 59-kDa protein bears exclusively flavin adenine dinucleotide in its oxidized state. Illumination with blue light induces formation of a neutral flavin radical with absorption maxima at 540 and 580 nm. The reaction proceeds aerobically even in the absence of an exogenous electron donor, which suggests that it reflects a physiological response. The process is completely reversible in the dark and exhibits a decay time constant of 200 s in the presence of oxygen. Binding of ATP strongly stabilizes the radical state after illumination and impedes the dark recovery. Thus, ATP binding has functional significance for plant cryptochromes and does not merely result from structural homology to DNA photolyase. The light-sensitive domain responds to illumination by an increase in phosphorylation. The autophosphorylation takes place although the protein is lacking its native C-terminal extension. This finding indicates that the extension is dispensable for autophosphorylation, despite the role it has been assigned in mediating signal transduction in Arabidopsis

Primary events in the blue light sensor plant cryptochrome: intraprotein electron and proton transfer revealed by femtosecond spectroscopy

Immeln D, Weigel A, Kottke T, Pérez Lustres JL. Primary events in the blue light sensor plant cryptochrome: intraprotein electron and proton transfer revealed by femtosecond spectroscopy. Journal of the American Chemical Society. 2012;134(30):12536-12546.Photoreceptors are chromoproteins that undergo fast conversion from dark to signaling states upon light absorption by the chromophore. The signaling state starts signal transduction in vivo and elicits a biological response. Therefore, photoreceptors are ideally suited for the analysis of protein activation by time-resolved spectroscopy. We focus on plant cryptochromes which are blue light sensors regulating the development and daily rhythm of plants. The signaling state of these flavoproteins is the neutral radical of the flavin chromophore. It forms on the microsecond timescale after light absorption by the oxidized state. We apply here femtosecond broadband transient absorption to early stages of signaling-state formation in an algal plant cryptochrome. Transient spectra show: i) sub-ps decay of flavin stimulated emission and ii) further decay of signal until 100 ps delay with nearly constant spectral shape.i) monitors electron transfer from a nearby tryptophan to the flavin and occurs with a time constant of τ(ET)=0.4 ps. ii) is analyzed by spectral decomposition and occurs with a characteristic time constant τ(1)=31 ps. We reason that hole transport through a tryptophan triad to the protein surface and partial deprotonation of tryptophan cation radical hide behind τ(1). These processes are probably governed by vibrational cooling. Spectral decomposition is used together with anisotropy to obtain the relative orientation of flavin and the final electron donor. This narrows the number of possible electron donors down to two tryptophans. Structural analysis suggests that a set of histidines surrounding the terminal tryptophan may act as proton acceptor and thereby stabilize the radical pair on a 100 ps timescale

Infrared spectrum and absorption coefficient of the cofactor flavin in water

Spexard M, Immeln D, Thöing C, Kottke T. Infrared spectrum and absorption coefficient of the cofactor flavin in water. VIBRATIONAL SPECTROSCOPY. 2011;57(2):282-287

Microsecond Light-induced Proton Transfer to Flavin in the Blue Light Sensor Plant Cryptochrome

Langenbacher T, Immeln D, Dick B, Kottke T. Microsecond Light-induced Proton Transfer to Flavin in the Blue Light Sensor Plant Cryptochrome. JOURNAL OF THE AMERICAN CHEMICAL SOCIETY. 2009;131(40):14274-14280

Photoreaction of Plant and DASH Cryptochromes Probed by Infrared Spectroscopy: The Neutral Radical State of Flavoproteins

Immeln D, Pokorny R, Herman E, Moldt J, Batschauer A, Kottke T. Photoreaction of Plant and DASH Cryptochromes Probed by Infrared Spectroscopy: The Neutral Radical State of Flavoproteins. Journal of Physical Chemistry B. 2010;114(51):17155-17161